1. Field of the Invention
This invention is directed to methods for preventing the emergence of multidrug resistance in tumor cells during cancer chemotherapy. In particular, it relates to the use of inhibitors of particular pathways of signal transduction to prevent the induction of the multidrug resistance (MDR1) gene by chemotherapeutic drugs. MDR1 gene expression, which results in tumor cell resistance to subsequent treatment with certain chemotherapeutic drugs is shown herein to be induced in response to treatment with various cytotoxic agents. Inhibitors of protein kinases, cytoplasmic calcium antagonists and calmodulin inhibitors, phosphoinositol-dependent phospholipase C inhibitors, and substances that inhibit activation of the transcription factor NF-.kappa.B (each of which have been implicated in intracellular eukaryotic signal transduction) are also shown herein to suppress this cellular response. Therefore, such inhibitors are useful in preventing MDR1 induction by chemotherapeutic drugs in a variety of tumor cells, when administered prior to and/or simultaneously with cytotoxic drug treatment in cancer patients.
2. Summary of the Related Art
Chemotherapy is a primary form of conventional cancer treatment. However, a major problem associated with cancer chemotherapy is the ability of tumor cells to develop resistance to the cytotoxic effects of anti-cancer drugs during the course of treatment. It has been observed that tumor cells can become simultaneously resistant to several chemotherapeutic drugs with unrelated chemical structures and mechanisms of action. This phenomenon is referred to as multidrug resistance. The best documented and clinically relevant mechanism for multidrug resistance in tumor cells is correlated with the expression of P-glycoprotein, the product of the MDR1 gene.
P-glycoprotein is a broad specificity efflux pump located in the cell membrane, and functions by decreasing the intracellular accumulation of many lipophilic cytotoxic drugs, including some widely used anticancer agents such as anthracyclines, vinca alkaloids, epipodophyllotoxins, actinomycin D and taxol, thereby rendering cells resistant to these drugs (Pastan and Gottesman, 1991, Annu. Rev. Med. 42: 277-286; Roninson (ed.), 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, New York; Schinkel and Borst, 1991, Sem. Cancer Biol. 2: 213-226).
Human P-glycoprotein is expressed in several types of normal epithelial and endothelial tissues (Cordon-Cardo et al., 1990, J. Histochem. Cytochem. 38: 1277-1287; Thiebaut et al., 1989, Proc. Natl. Acad. Sci. USA 84: 7735-7738), as well as in hematopoietic stem cells (Chaudhary and Roninson, 1991, Cell 66: 85-94), and a subpopulation of mature lymphocytes (Neyfakh et al., 1989, Exp. Cell Res. 185: 496-505). More importantly, MDR1 mRNA or P-glycoprotein have been detected in most types of human tumors, both before and after chemotherapeutic treatment (Goldstein et al., 1989, J. Natl. Cancer Inst. 81: 116-124; Noonan et al., 1990, Proc. Natl. Acad. Sci. USA 87: 7160-7164). The highest levels of MDR1 expression are usually found in tumors derived from MDR1-expressing normal tissues; e.g., renal, adrenocortical or colorectal carcinomas. In other types of solid tumors and leukemias, MDR1 expression prior to treatment is usually relatively low or undetectable, but a substantial fraction of such malignancies express high levels of MDR1 after exposure to chemotherapy (Goldstein et al., 1989, ibid.). Prior to the present invention, the increase in MDR1 expression after chemotherapy was believed to result from in vivo selection for rare, pre-existing tumor cells that were already inherently resistant to chemotherapeutic drugs due to MDR1 expression.
Even low levels of MDR1 expression have been correlated with the lack of response to chemotherapy in several different types of cancer (Chan et al., 1990, J. Clin. Oncol. 8: 689-704; Chan et al., 1991, N. Engl. J. Med. 325: 1608-1614; Musto et al., 1991, Brit. J. Haematol. 77: 50-53), indicating that P-glycoprotein-mediated multidrug resistance represents an important component of clinical drug resistance. Whereas many clinical and pre-clinical studies have addressed pharmacological strategies for inhibiting P-glycoprotein function (Ford and Hait, 1990, Pharmacol. Rev. 42: 155-199), prior to the present invention, little was known about the factors that are responsible for the induction or up-regulation of P-glycoprotein expression in tumor cells under conditions relevant to cancer chemotherapy. Understanding such factors provides insight into the development of methods for preventing the appearance of P-glycoprotein in human tumors, thus reducing the incidence of multidrug resistance in cancer, and leading to more effective chemotherapy of cancer.
Numerous gene transfer studies have demonstrated that elevated expression of the MDR1 gene is sufficient to confer the multidrug resistance phenotype (Roninson, 1991, ibid.). For instance, mouse NIH 3T3 cells infected with a recombinant retrovirus carrying human MDR1 cDNA became multidrug-resistant in proportion to the density of human P-glycoprotein on their surface; the correlation was not affected by the presence or absence of cytotoxic selection (Choi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 7386-7390).
Nevertheless, consistent association of other biochemical changes with multidrug-resistant cells suggested that these alterations may also play a role in multidrug resistance, possibly by affecting the expression or function of P-glycoprotein. The most prominent of such changes is the increased activity of protein kinase C (PKC), found in many, but not all, multidrug-resistant cell lines obtained after multiple steps of cytotoxic selection (Aquino et al., 1990, Cancer Commun. 2: 243-247; Fine et al., 1988, Proc. Natl. Acad. Sci. USA 85: 582-586; O'Brian et al., 1989, FEBS Lett. 246: 78-82; Posada et al., 1989, Cancer Commun. 1: 285-292). PKC activation has been shown to increase the level of drug resistance in some drug-sensitive and multidrug-resistant cells lines (Ferguson and Cheng, 1987, Cancer Res. 47: 433-441; Fine et al., 1988, ibid.; Yu et al., 1991, Cancer Commun. 3: 181-189). Although PKC is reported to be capable of phosphorylating P-glycoprotein (Chambers et al., 1990, Biochem. Biophys. Res. Commun. 169: 253-259; Chambers et al., 1990, J. Biol. Chem. 265: 7679-7686; Hamada et al., 1987, Cancer Res. 47: 2860-2865), it is not known whether such phosphorylation is responsible for the observed changes in drug resistance. While it has been shown that certain PKC inhibitors reversed multidrug resistance in some P-glycoprotein expressing cell lines (O'Brian et al., 1989, ibid.; Posada et al., 1989, Cancer Commun. 1: 285-292; Palayoor et al., 1987, Biochem. Biophys. Res. Commun. 148: 718-725), the available evidence suggests that at least some of the observed effects were due to direct inhibition of P-glycoprotein function by the tested compounds rather than inhibition of PKC-mediated phosphorylation (Ford et al. 1990, Cancer Res. 50: 1748-1756; Sato et al., 1990, Biochem. Biophys. Res. Commun. 173: 1252-1257). These studies have provided no indication that PKC-interactive agents could have an effect on expression, rather than phosphorylation or function, of P-glycoprotein.
Several laboratories have investigated the factors that regulate MDR1 gene expression in normal and malignant cells. One example of normal physiological regulation of an MDR1 homolog was found in mouse uterine endometrium, where the expression of a mouse mdr gene was induced by steroid hormones at the onset of pregnancy (Arceci et al., 1990, Molec. Repro. Dev. 25: 101-109; Bates et al., 1989, Molec. Cell. Biol. 9: 4337-4344). In rat liver, the expression of an mdr gene was found to be inducible by several carcinogenic or cytotoxic xenobiotics; similar induction was also observed during liver regeneration (Fairchild et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7701-7705; Thorgeirsson et al., 1987, Science 236: 1120-1122). Further, a rodent homolog of MDR1 was induced in several cell lines in response to treatment with certain cytotoxic drugs (Chin et al., 1990, Cell Growth Diff. 1: 361-365). In contrast, no induction of the human MDR1 gene by cytotoxic drugs was detected in any of the human cell lines tested in the same study. Other investigators have also failed to detect MDR1 induction upon treatment with cytotoxic drugs (Schinkel and Borst, 1991, Sem. Cancer Biol. 2: 213-226).
Several studies have indicated, however, that the human MDR1 gene may be susceptible to stress induction, under certain conditions. Thus MDR1 expression in some human cell lines was increased by treatment with heat shock, arsenite (Chin et al., 1990, J. Biol. Chem. 265: 221-226) or certain differentiating agents (Mickley et al., 1989, J. Biol. Chem. 264: 18031-18040; Bates et al., ibid.). Some cytotoxic P-glycoprotein substrates were reported to stimulate transcription of a reporter gene from the human MDR1 promoter (Kohno et al., 1989, Biochem. Biophys. Res. Commun. 165: 1415-1421; Tanimura et al., 1992, Biochem. Biophys. Res. Commun. 183: 917-924) and to increase P-glycoprotein expression in a mesothelioma cell line after prolonged exposure (Light et al., 1991, Int. J. Cancer 49: 630-637). Despite such reports of MDR1 induction, however, it has never been determined whether short-term exposure to any agents used in cancer chemotherapy could induce expression of the MDR1 gene in human cells, and whether MDR1 induction could be prevented.
Recently, Kiowa et al. (1992, FEBS Lett. 301: 307-309) have reported that the addition of a flavonoid, quercetin, can prevent an increase in MDR1 expression in a hepatocarcinoma cell line induced by arsenite, a compound which is not used in cancer treatment, but is known to activate the transcriptional pathway mediated by the heat shock response element in the MDR1 promoter. Although not disclosed by Kiowa et al., inhibition of PKC activity is one of the biological effects of quercetin (Gschwendt et al., 1984, Biochem. Biophys. Res. Commun. 124: 63). It is possible therefore that PKC inhibition by quercetin could be responsible, in part, for the observed inhibition of MDR1 induction by arsenite. However, it is noteworthy that the ability of quercetin to inhibit a transcriptional response mediated by the heat shock response element is believed to those skilled in the art to be unrelated to PKC inhibition. (see, e.g., Kantengwa and Polla, 1991, Biochem. Biophys. Res. Commun. 180: 308-314). Furthermore, Kiowa et al. provide no suggestion that non-flavonoid PKC inhibitors would be able to inhibit MDR1 induction by arsenite, or that quercetin would be able to inhibit the induction of MDR1 expression when used in combination with chemotherapeutic drugs or any other agents that are not known to activate the heat shock response element-mediated pathway.
In addition to MDR1, another pleiotropic drug transporter has been recently discovered (Grant et al., 1994, Cancer Res. 54: 357-361)). This protein, termed the Multidrug Resistance-associated Protein (MRP), has been shown to confer a pattern of resistance to cytotoxic, particularly chemotherapeutic, drugs similar to the P-glycoprotein transporter encoded by the MDR1 gene. No inhibitors of MRP expression have been previously reported.